The PI discovered the first metastasis suppressor gene, Nm23 (NME). Metastasis suppressor genes are genes that, when re-introduced into metastatic tumor cells at physiologic levels of expression, significantly inhibit metastasis in vivo without affecting primary tumor size. These genes provide a window on the metastatic process independent of events in the primary tumor. Many metastasis suppressor genes have now been described and our goal is to translate this knowledge to the clinic. In my lab, basic and translational research has investigated the role of NME1 in the regulation of tumor metastasis. Eleven transfection studies have documented that overexpression of NME1 or NME2 in various tumor cell lines resulted in a 50-90% decrease in tumor metastatic potential in vivo. The mechanism of Nm23 suppression of metastasis is incompletely understood and likely complex. In the past year we have conducted two fundamental mechanistic studies: (1) We returned to the histidine protein kinase activity of NME1. NME1 has been reported to be a histidine protein kinase but the contribution of this function has been impossible to determine, as histidine is destroyed under acidic and heat conditions used for simple SDS-PAGE. Based on a recent publication of an anti- phospho-histidine monoclonal antibody, we have been able to quantify and visualize NME-pHis for the first time. Using sets of triple-negative breast cancer cell lines expressing a vector or NME1, total NME1 and pHis-NME1 levels were often directly correlated. NME1 mutants were overexpressed in breast cancer cells that separated the NDP kinase and histidine protein kinase activities of NME1. In these mutants, the histidine protein kinase expression of NME1 best correlated with inhibition of breast cancer motility in vitro. The data suggest that the histidine protein kinase activity of NME may be important for its metastasis suppression. (2) The lab also investigated the role of dynamin 2 (DNM2) in NME function. The role of dynamin (DNM2), a GTPase regulating membrane scission of vesicles in endocytosis, was examined in NME1 and NME2 regulation of tumor cell motility and metastasis. Overexpression of NMEs in two cancer cell lines increased endocytosis of transferrin receptor (TfR) and EGF receptor (EGFR) concurrent with motility and migration suppression; internalized vesicles costained with Rab5, depleted AP2 from cell surface, and exhibited increased Rab5-GTP levels, consistent with endocytosis. The dynamin inhibitors Iminodyne-22 and Dynole-34-2, or shRNA-mediated downregulation of DNM2, impaired NME ability to augment endocytosis or suppress tumor cell motility. In a lung metastasis assay NME1 overexpression failed to significantly suppress metastasis in the DNM2 knockdown cells. Using the EGF-EGFR signaling axis as a model in MDA-MB-231T cells, NME1 decreased pEGFR and pAkt expression in a DNM2-dependent manner, indicating the relevance of this interaction to downstream signaling. NME/DNM2 interaction was confirmed in two-way coimmunoprecipitations. Using transfection of a NME1 site directed mutant lacking a histidine protein kinase activity but retaining a nucleoside diphosphate kinase (NDPK) activity, the NDPK activity of NME was insufficient to promote endocytosis or inhibit EGFR signaling. We report a novel mechanism of action: Addition of NME1 or NME2 to DNM2 in vitro facilitated DNM2 oligomerization and increased GTPase activity, both required for vesicle scission. A NMEDNM2 interaction may contribute to metastasis suppression by altering the tumor endocytic and motility phenotypes.